Figure 8.

K5 Expression Reduces Targeting by NK Cells

(A) Depiction of targeting of NK-activating ligands, such as MIC-A, for ubiquitination and endocytosis by K5. (B) Flow cytometry identifying K562 cells transduced to express Qstalk alone or coexpressing K3 or K5. These K562 populations were cotransduced to express an eGFP-firefly luciferase fusion reporter (see Figure S4C). (C) Transduced K562 cells were cocultured with freshly isolated human peripheral blood NK cells at decreasing E:T ratios for 24 h. Specific lysis was calculated in relation to the average level of luciferase activity of NT K562 cells at an E:T of 10:1. Multiple t tests were used to compare the difference between the mock- and K5-modified group. ∗p < 0.05; ∗∗p < 0.01. (D) Mixed populations of transduced (marked with CD34Q) and NT K562 cells were cocultured with NK cells for 3 days. Viability was determined by CMFDA exclusion. (E and F) Plot of loss of viability of the K562 cell expressing Qstalk alone or K5-Qstalk against a decreasing number of NK cells. (F) Killing of Qstalk-K562 cells within the same experimental populations as (E). Two-way ANOVA was used to determine the differences among groups, p < 0.01 (E); p = 0.68 (F).